Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

Abstract

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-M range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower highmass sensitivity of MCP detectors. Several major effects have been described to result in nonnative conditions for the noncovalent complexes: MALDI matrix [3][4][5], sample preparation [4,6,7], crystal morphology [4,8,9], pH of the solution [6, 10 -12], organic solvent [7,8], ionic strength [13,14], matrix/analyte ratio [15,16], speed of solvent evaporation [4], and sample concentration [9,15]. The effect of some instrumental parameters in the detection of complexes has also been studied; extraction delay time, positive/negative ion mode, linear/reflector and acceleration mode were found to be of minor importance [7,17], whereas laser pulse energy [8,17,18] and the number of laser shots (i.e., "first shot phenomenon") [4,8,13,19,20] was reported to be a decisive factor in a number of cases.Also, analyses by electrospray ionization (ESI) [21] are not carried out at physiological conditions as only solutions of very low ionic strength can be analyzed. However, ESI generates "colder" ions than MALDI and it keeps the sample in aqueous "biological" environment before ionization. For these reasons, ESI has been used in numerous studies to detect noncovalent complexes [22].Relatively few cases have been reported where specific intact noncovalent complexes were successfully observed with MALDI. Besides the frequent dissociation of noncovalent complexes due to the experimental conditions employed, a further...

Indirect study of non‐covalent protein complexes by MALDI mass spectrometry: Origins, advantages, and applications of the “intensity‐fading” approach

“…Similar conclusions were reached in the study of protease inhibitor complexes (Yanes et al, ). The investigation of the effects of different matrix solutions at different pH, with and without the use of thin layer deposition, on the stability of two protease inhibitor complexes, and thus the relative intensity of the ligand, was largely unaffected by pH in the range of 2.5–5.5 (Yanes et al, ) but could be disrupted below this pH range.…”

Investigation of calmodulin-peptide interactions using matrix-assisted laser desorption/ionization mass spectrometry

“…Recent studies have suggested that the specific interactions between proteins and their peptide ligands could be deduced using IF-MALDI-MS, namely, introduction of a protein receptor could result in a selective decrease of the ion abundances of its peptide ligands compared with the nonbinding control [25][26][27][28][29][30][31][32][33][34].…”

“…When the signal intensity reached the saturation point, further increase of the laser intensity made the fading phenomena less pronounced and it was difficult to discriminate the target peptides with the nonbinding control. Besides these instrumental parameters, the influence of analyte concentration on the fading phenomena was also investigated because previous studies suggested that the analyte concentration might have an impact on the observed fading [34]. Peptide mixtures containing the target peptide at desired concentrations were incubated with equal amount of calcium-saturated calmodulin (1:1 M), followed by the addition of matrix solutions.…”

“…Recently, Aviles and coworkers have developed an alternative strategy to detect the presence of noncovalent complexes in solution using intensityfading (IF-) MALDI-MS [25][26][27], which was based on a selective reduction of the ion abundance of the specific ligands after the addition of their receptor protein. The strategy was similar to the immunological procedures for identification of the epitopic peptides for antibodies introduced by the Downard group [28 -31] and has shown promising applications to detect several biological interactions [25][26][27][28][29][30][31][32][33][34][35][36][37]. Nevertheless, it should be noted that a nonbinding control is usually necessary to assess the specificity of binding assay in both direct MALDI-MS and IF-MALDI MS analysis [24 -37].…”

A novel, simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry

“…The detection of non‐covalent interactions was demonstrated a few years after the introduction of MALDI MS,5 e.g. antibody–antigen interactions;5–9 intensity fading where the ion intensity from a ligand is suppressed in the presence of a binding protein;10, 11 three‐dimensional biochip fabrication using methacrylate polymers, where proteins are immobilized followed by incubation with appropriate ligands9 and desorption/ionization on porous silica (DIOS), which has been used for functional characterization and identification of small biomolecules without use of matrix 12, 13. The general applicability of these methods is dependent on ease of use and sensitivity.…”